Association of citrullinated proteins with synovial exosomes.

OBJECTIVE: In addition to releasing proteins and mediators, cells also release membrane vesicles (exosomes and apoptotic blebs) into the extracellular environment. Apoptotic blebs contain multiple autoantigens, but few data are available concerning the protein content of exosomes. Exosomes are formed during an immune response and can directly stimulate T cells or bind to dendritic cells. The aim of this study was to identify the nature of synovial exosomes from patients with different rheumatic diseases and to examine their potential autoantigenic content, which may be involved in the induction of an autoimmune response. METHODS: Synovial exosomes from patients with rheumatoid arthritis (RA), patients with reactive arthritis, and patients with osteoarthritis were purified, analyzed by electron microscopy, and labeled with immunogold to detect IgG and IgM molecules. Autoantigen content was identified by 2-dimensional electrophoresis-immunoblotting and subsequent mass spectrometry. In order to investigate the presence of citrullinated proteins, immunoblotting with anticitrulline antibodies was performed. RESULTS: Citrullinated proteins were observed in all exosome preparations, in contrast to other autoantigenic proteins (e.g., BiP and heterogeneous nuclear RNP A2) that were previously observed in RA and other autoimmune diseases. These citrullinated proteins included the fibrin alpha-chain fragment, fibrin beta-chain, fibrinogen beta-chain precursor, fibrinogen D fragment, and the Sp alpha (CD5 antigen-like protein) receptor. Purification of synovial exosomes led to the detection of citrullinated fibrinogen and citrullinated Sp alpha associated with IgM and IgG. CONCLUSION: Synovial exosomes contain citrullinated proteins, which are known to be autoantigens in RA. Although immune mechanisms in which exosomes carry citrullinated peptides could play an important role in the induction and distribution of citrullinated proteins, there must be a specific recognition of these proteins that is unique to the RA immune system.

FOXP3 identifies regulatory CD25bright CD4+ T cells in rheumatic joints.

Regulatory T cells have recently been implicated in a number of human diseases, including rheumatoid arthritis. To investigate whether the presence of CD25+CD4+ regulatory T cells is a general finding in arthritic joints, synovial fluid of patients with different rheumatic diseases such as undifferentiated arthritides, systemic rheumatic diseases and reactive arthritis were investigated for the presence of such cells. In 95% of the patients, a higher frequency of CD25(bright)CD4+ T cells was found in synovial fluid as compared with peripheral blood. Both in vitro suppression experiments and FOXP3 mRNA analysis confirmed these cells to be natural regulatory T cells. Together with our previous data, we conclude that arthritic joints, irrespective of precise diagnosis and disease duration, are enriched with natural regulatory T cells. These results suggest that suppressor cells migrate to and/or multiply at the sites of inflammation as part of the immune responses' effort to combat injurious inflammation.

Chemokine and chemokine receptor expression in paired peripheral blood mononuclear cells and synovial tissue of patients with rheumatoid arthritis, osteoarthritis, and reactive arthritis.

BACKGROUND: Chemokine receptors and chemokines have a crucial role in leucocyte recruitment into inflamed tissue. OBJECTIVE: To examine the expression of an extensive number of chemokines and receptors in a unique bank of paired samples of synovial tissue (ST) and peripheral blood (PB) from patients with different forms of arthritis to assist in identifying suitable targets for therapeutic intervention. METHODS: Synovial biopsy specimens were obtained from 23 patients with rheumatoid arthritis (RA), 16 with osteoarthritis, and 8 with reactive arthritis. ST chemokine (CCL2/MCP-1, CCL5/RANTES, CCL7/MCP-3, CCL8/MCP-2, CCL14/HCC-1, CCL15/HCC-2, CCL16/HCC-4), chemokine receptor (CCR1, CCR2b, CCR5, CXCR4), and CD13 expression was analysed by immunohistochemistry and two colour immunofluorescence. Chemokine receptor expression (CCR1, CCR3, CCR5, CCR6, CCR7) on PB cells was studied by flow cytometry. Non-parametric tests were used for statistical analysis. RESULTS: Abundant expression of CCR1, CXCR4, and CCR5 was found in all forms of arthritis, with a specific increase of CCL5 and CCL15 in RA. CCL7, CCL8, CCL14, CCL15, and CCL16 were detected for the first time in ST. The results for PB analysis were comparable among different arthritides. Interestingly, compared with healthy controls, significantly lower expression of CCR1 (p<0.005) and CCR5 (p<0.05) by PB monocytes in the patient groups was seen. DISCUSSION: A variety of chemokines and receptors might have an important role in several inflammatory joint disorders. Although other receptors are involved as well, migration of CCR1(+) and CCR5(+) cells towards the synovial compartment may play a part in the effector phase of various forms of arthritis.

Bacterial components in the synovial tissue of patients with advanced rheumatoid arthritis or osteoarthritis: analysis with gas chromatography-mass spectrometry and pan-bacterial polymerase chain reaction.

OBJECTIVE: To study the presence of bacterial components in the synovial tissue (ST) of patients with advanced rheumatoid arthritis (RA). METHODS: ST was collected during joint surgery from 41 RA patients. Tissue from 39 patients with osteoarthritis (OA), 4 patients with undifferentiated inflammatory arthritis (UA), and 3 cases of accidental deaths served as controls. The pan-bacterial polymerase chain reaction (PCR) with primers for the 23S ribosomal RNA (rRNA) and 16S rRNA genes was used to detect bacterial DNA. In addition, synovial fluid (SF) samples from patients with chlamydial reactive arthritis (ReA) were also examined by the same method. The positive controls, bacterial DNA or ST spiked with different living bacteria, were analyzed alongside clinical samples. Most of the ST samples were also analyzed by gas chromatography-mass spectrometry (GC-MS) for determining the presence of bacteria-derived muramic acid. Strict precautions were followed in the clinics and the laboratory to prevent contamination. RESULTS: In GC-MS analysis, muramic acid was observed in the ST from 4 of 35 RA patients and from 2 of 14 OA patients, but not in ST from 2 patients with UA and 3 cadavers. Bacterial DNA was not detected by either one of the PCR primers used in ST from 42 patients with RA and 39 patients with OA. However, 5 of 15 SF samples from ReA patients were PCR positive. The sensitivity of GC-MS to detect muramic acid was 2 pg/injected amount (227 pg muramic acid/mg ST), and that of the pan-bacterial PCR was 2-20 bacteria colony forming units/reaction. CONCLUSION: These results indicate that a bacterial component, muramic acid, is detectable by GC-MS in ST from a few patients with advanced RA or OA. However, no bacterial DNA was detectable by PCR.

[Lipid peroxidation abnormalities in miners with Reiter's disease and gout]. / Narushenie perekisnogo okisleniia lipidov u shakhterov bol'nykh bolezn'iu Reitera i podagroi.

The paper presents results of investigations designed to study changes in lipid peroxidation and in antioxidant system of miners in different vocational groups with Reiter's disease and gout. It is shown that the condition of the above systems is affected by both diseases as such and occupational conditions of miners. Based on the secured findings it is recommended that treatment of the above patients be supplemented with an antioxidant agent, copherol in particular.

Excitatory amino acid profiles of synovial fluid from patients with arthritis.

OBJECTIVE: Previous studies in an experimental synovitis model in rats determined that administration of glutamate and aspartate into the joint produces hyperalgesic responses, while their receptor antagonists provide protection against the development of a hyperalgesic state. We examined concentrations of amino acids in synovial fluid (SF) to determine if increases might be relevant to human joint pathology. METHODS: One hundred forty-four repository SF samples from patients undergoing diagnostic or therapeutic arthrocentesis and 14 SF samples from 7 cadavers were analyzed by high pressure liquid chromatography and compared as arthritic and control cohorts. RESULTS: Compared to the average concentrations from the autopsy cases, the excitatory amino acids (EAA) glutamate and aspartate in SF from patients with synovitis were 54 and 28 times higher, respectively. Increases for all other amino acids ranged from 3 to 18-fold. The values for glutamate and aspartate were significantly higher than the mean increase for other amino acids compared using unpaired t tests (p < 0.0001). The mean ratio of glutamate and aspartate elevations over the mean increase for other amino acids was 4-fold and 2-fold, respectively. The EAA were highest in Reiter's, infectious arthropathies, and systemic lupus erythematosus, but did not appreciably segregate to diagnosis or SF white blood cell count. CONCLUSION: Our data provide evidence of increased glutamate and aspartate in the SF of humans with active arthritis, suggesting that glutamate mediated events may contribute to the pathogenesis of human arthritic conditions.

Expression of adhesion molecules on synovial fluid and peripheral blood monocytes in patients with inflammatory joint disease and osteoarthritis.

OBJECTIVE: To determine the presence of adhesion molecules on monocytes/macrophages (Mphi) from peripheral blood (PB) and synovial fluid (SF) in patients with osteoarthritis (OA) and inflammatory joint diseases (rheumatoid (RA) and reactive arthritis (ReA)) in order to improve our understanding of the possible mechanisms underlying the inflammatory process. METHODS: Whole blood and SF cells were stained with monoclonal antibodies against CD11a (LFA-1), CD15 s (sialyl-Lewis X), CD44, CD54, VLA-4, and HLA-DR counterstained with anti-CD14 antibodies as a Mphi marker for dual fluorescence analysis by flowcytometry. RESULTS: On PB-Mphi, CD15s was markedly increased in both RA as well as ReA compared with OA. Furthermore, in the PB LFA-1, CD44, and HLA-DR showed a higher surface density on Mphi in ReA than in OA. Comparison between SF and PB showed significantly higher CD44 and CD54 expression on SF-Mphi. These molecules play an important part in lymphocyte-Mphi interaction. CONCLUSION: In PB from patients with inflammatory joint diseases, Mphi are activated, allowing recruitment into the synovial compartment. These disorders, in contrast with OA seem to be "systemic" in nature. Within the SF, different adhesion molecules are expressed on CD14(+) Mphi as compared with PB.

Elevated nerve growth factor levels in the synovial fluid of patients with inflammatory joint disease.

A novel pH shock extraction procedure was used to measure nerve growth factor (NGF) levels in both normal and inflamed synovial fluids using a sensitive and specific two-site enzyme linked immunosorbant assay. To date no data is available on NGF levels in normal synovial fluids. Synovial fluids were taken from 5 normal volunteers, 12 patients with rheumatoid arthritis and 10 patients with other inflammatory arthropathies. The mean +/- SEM NGF concentration in normal synovial fluids was 95 +/- 33.2 pg/ml (range 39.1-143.1 pg/ml), whereas the mean NGF concentration in the synovial fluids taken from patients with rheumatoid arthritis was 532.5 +/- 123.8 pg/ml (range 152-1686 pg/ml). The mean NGF concentration in patients with other inflammatory arthropathies was also raised (430.6 +/- 90 pg/ml; range 89-1071 pg/ml). The NGF concentrations were significantly higher in the synovial fluids from both inflamed groups (ANOVA p < 0.05) compared to normals. Raised levels of NGF in synovial fluid may contribute directly to joint inflammation via activation of inflammatory cells.

Detection of bacterial DNA in joint samples from patients with undifferentiated arthritis and reactive arthritis, using polymerase chain reaction with universal 16S ribosomal RNA primers.

OBJECTIVE: Bacteria are considered to be important in the pathogenesis of several forms of arthritis. The goal of this study was to apply the 16S ribosomal RNA (rRNA)-polymerase chain reaction method for the detection of bacterial DNA in synovial fluid (SF) and synovial tissue (ST) from inflamed joints. METHODS: Samples from 5 patients with septic arthritis and from 7 with osteoarthritis or arthritis secondary to joint trauma were used as controls. Samples from 6 patients with spondylarthropathy (SpA) and from 20 with undifferentiated arthritis (UA) were analyzed for the presence of bacterial DNA using universal 16S rRNA primers. Automated sequencing and comparative data analysis were performed to identify the species. RESULTS: In the positive control group, the bacterial species cultured from the synovium could be identified in all cases. No bacterial DNA was detected in the SF and ST from patients in the negative control group. In 4 of 6 patients with SpA and 7 of 20 with UA, the analysis of joint samples revealed the presence of bacterial DNA. Sequence analysis indicated the presence of multiple species, which was confirmed by sequencing of cloned products. CONCLUSION: When the the above techniques were used with a stringent regimen, we were able to demonstrate that it is possible to collect and analyze joint samples without contaminating bacterial DNA. The accumulation of phagocytic cells that contain bacterial DNA of various species could play a role in the pathogenesis of both SpA and UA.

Measurement of synovial tumor necrosis factor-alpha in diagnosing emergency patients with bacterial arthritis.

Because of the high morbidity and mortality in patients with bacterial arthritis, rapidly and correctly diagnosing this critical condition is a challenge to emergency clinicians. Synovial fluid samples were obtained from 75 patients with arthritis disorders who presented to an emergency service, and levels of tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), and interleukin-6 (IL-6) were measured. Twenty patients with culture-proven bacterial arthritis had higher levels of synovial TNF-alpha than patients with osteoarthritis or with inflammatory arthritis, including gouty arthritis, rheumatoid arthritis, reactive arthritis, and lupus arthritis. There was a good sensitivity for synovial TNF-alpha level in diagnosing patients with bacterial arthritis. Nearly 100% of patients with bacterial arthritis had elevated synovial TNF-alpha levels. However, synovial IL-1 beta and IL-6 levels failed to discriminate bacterial arthritis from other inflammatory arthritis. Measurement of synovial TNF-alpha level may be useful as a diagnostic aid in emergency patients with bacterial arthritis disorders.

Oncoprotein expression in human synovial tissue: an immunohistochemical study of different types of arthritis.

Based on the fact that synovial lining cells have some properties of transformed-appearing cells, we have examined the expression of Myc, Myb, Fos, Jun and Ras oncoproteins in synovial tissues from patients with different types of arthritis. Formalin-fixed and paraffin-embedded sections of synovial tissue from 12 patients with rheumatoid arthritis (RA), 14 with reactive arthritis (ReA), nine with other seronegative arthritis (OSA), seven with bacterial arthritis (BA), eight with probable bacterial arthritis (PBA) and eight with osteoarthritis (OA) were studied using the immunoperoxidase staining technique. The oncoproteins studied were expressed both in the synovial lining layer and in the sublining layer, consisting of lymphocytes, other inflammatory cells and blood vessels. Among the six disease entities, RA and OA appeared to be the most distinct, whereas the results obtained for ReA and OSA, and on the other hand for BA and PBA, closely resembled each other. The expression of Myc, Myb, Fos and Jun was significantly correlated both to the degree of synovial hypercellularity and the synovial lymphocytic infiltration. For Ras, such a correlation could not be seen. We conclude that we find no evidence of a cell lineage-specific or a disease-specific abnormality of proto-oncogene products in RA, and the expression of these oncoproteins is consistent with inflammation rather than with any primary abnormality of cell growth.

[Blood serum and synovial fluid proteins in Reiter's syndrome]. / Belki syvorotki krovi i sinovial'noi zhidkosti pri sindrome Reitera.

Individual proteins (transferrin, ceruloplasmin, alpha 2-macroglobulin, IgG, IgA and IgM) were examined in blood and synovial fluid of 11 patients with Reiter's syndrome. ARA-1981 was used as a diagnostic criteria. The control group included 40 patients with rheumatoid arthritis and 31 with osteoarthrosis of the knee joint. Statistically significant differences between the concentrations of individual proteins in patients with Reiter's syndrome, rheumatoid arthritis and osteoarthritis were only established in synovial fluid for IgM and for IgM index Csf/CS.

Substance P and arthritis: analysis of plasma and synovial fluid levels.

The uncadecapeptide substance P (SP), which is localized in peripheral and central terminals of afferent nerve fibers with polymodal nociceptors, has recently been implicated as having a neurogenic, inflammatory role in experimental arthritis. We used a radioimmunoassay to measure SP levels in plasma and synovial fluid samples from patients with rheumatoid arthritis (RA), osteoarthritis (OA), Reiter's syndrome (RS), and posttraumatic arthritis, as well as in plasma samples from 13 normal subjects. Plasma SP levels in RS patients exceeded levels in RA and OA patients, which in turn exceeded levels in posttrauma patients and in normal subjects. Synovial fluid SP levels exceeded respective plasma levels for all groups, except in RS patients, in whom the plasma level was not significantly different from that in synovial fluid. SP levels in synovial fluid of RA, OA, and RS patients did not differ significantly from each other, but the level in posttrauma patients was higher than in all other groups (P less than 0.005). These studies demonstrate localized intraarticular SP release, and significant plasma/synovial fluid SP concentration gradients in several forms of arthritis.

Interleukin 2 and interleukin 2 inhibitors in human serum and synovial fluid. II. Mitogenic stimulation, interleukin 2 production and interleukin 2 receptor expression in rheumatoid arthritis, psoriatic arthritis and Reiter's syndrome.

Peripheral blood and synovial fluid (SF) mononuclear cells from 30 patients with rheumatoid arthritis (RA) were hyporesponsive to mitogenic stimulation with plant lectins and CD3 antibodies, due to depressed interleukin 2 (IL-2) production and IL-2 receptor upregulation. In contrast, in the seronegative arthritis patient group only SF mononuclear cells were hyporesponsive to mitogenic stimulation and there were no significant differences in IL-2 production or IL-2 receptor upregulation as compared with control subjects. No significant correlations were observed between IL-2 inhibitor levels and mitogenic responses, IL-2 production and IL-2 receptor upregulation on peripheral blood and SF mononuclear cells but an inverse correlation was noted between SF mitogenic responses and the expression of selected activation markers. We conclude that IL-2 abnormalities appear to be most pronounced in RA compared with other inflammatory arthritides and that these changes do not appear to be directly related to serum or SF IL-2 inhibitor levels.

Synovial fluid lactate levels in septic and non-septic arthritides.

Lactate concentration was studied in 383 synovial fluid specimens from patients with various arthritides. The highest concentrations of lactate occurred in non-gonococcal septic synovial fluids. High values were recorded in seropositive rheumatoid arthritis and crystal-induced arthritides, medium values in synovial fluids from seronegative rheumatoid arthritis, seronegative spondylarthritides, gonococcal arthritis and haemarthrosis, and the lowest values in aspirates from osteoarthrotic joints. There was a positive correlation between synovial pH and lactic acid concentration. These data suggest that determination of lactate in synovial fluid can be valuable in the rapid exclusion of septic arthritis. Its value for differentiating between other inflammatory arthritides is discussed.

Fibronectin in the synovium of chronic inflammatory joint disease.

The localisation of fibronectin in the synovial membrane of rheumatoid arthritis (RA) and other chronic inflammatory joint diseases has been studied using a peroxidase-antiperoxidase immunohistochemical method. Synovia were studied from seven cases of seropositive RA three cases of seronegative RA, six cases of ankylosing spondylitis, four cases of Reiter's syndrome and five of psoriatic arthritis. Six were small biopsies and the remaining tissues were obtained at open surgery for orthopaedic procedures or biopsies. Fibronectin was demonstrated in all of the synovia examined and was present in intimal cells, synovial giant cells, the walls of small blood vessels, basement membrane of larger vessels and deposits of fibrin. No difference in this distribution of fibronectin was found in seropositive and seronegative RA, ankylosing spondylitis, Reiter's syndrome or psoriatic arthritis, neither was there any difference in the amount of fibronectin at various sites.